MitoEAGLE Short-Term Scientific Mission

My name is Beata Velika and I work as Research specialist at the Pavol Jozef Safarik University in Kosice, Department of Medical and Clinical Biochemistry, Faculty of Medicine, Slovakia. During my study and work I specialized in fluorescence spectroscopy, and I have experience with different isolation methods, including mitochondria isolation, and measuring of their native fluorescence and respiration.

I would like to carry out the project in cooperation with the research group at the Mitochondrial Research Laboratory of Prof. Dr. Erich Gnaiger in Innsbruck, Austria (Medical University of Innsbruck; K-Regio project MitoFit) in collaboration with the scientific company OROBOROS INSTRUMENTS Corp. The laboratory of Prof. Dr. Erich Gnaiger contains very high standard laboratory equipment, with several specialists in mitochondrial physiology field. The short-term project is focused on the optimization of SUIT (substrate-uncoupler-inhibitor titration) protocols for high-resolution respirometry of intact cryopreserved human peripheral blood mononuclear cells (PBMC). The usage of human PBMC will provide a significant stimulus for the entire field of mitochondrial medicine, because the mitochondrial function of PBMC is linked with numerous diseases and metabolic conditions in the human body, and the protocols developed during the project will lead to the standardization of the procedures, and should be used for studying mitochondria physiological and pathological mechanisms. Cryopreserved cells (stored at -80 °C), will be thawed in 37 °C water bath, dilluted in 10 ml pre-warmed 37 °C medium composed of DPBS + 2%FBS and centrifuged (300 g, 6 minutes). The sediment will be resuspended and counted. The suspension of cryopreserved cells will be titrated into the 2-ml chambers of the Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria). We will optimize the SUIT protocols with defined substrate combinations, uncoupler and inhibitor titrations, which will be applied for the measurement of respiration of the cryopreserved cells. The results will be compared with the respiration of the fresh cells (reference). Obtained results will be presented in 10th MiPschool 2017 MitoEAGLE and MitoEAGLE Workshop WG1-4, Obergurgl AT (2017 Jul 23-30).